Broadly neutralizing antibodies to sars-cov-2 and uses thereof

Abstract

The present application relates to sarbecovirus broad-spectrum neutralizing antibodies and applications thereof. The sarbecovirus broad-spectrum neutralizing antibodies of the present application can bind to sarbecovirus S proteins in a broad spectrum, including but not limited to SARS-CoV-2 and its variant strains, SARS-CoV, WIV1, GD / 1 / 2019, GX / P2V / 2017, RaTG13, etc., and neutralize the infection of SARS-CoV-2 and its variant strains, SARS-CoV, WIV1, GD / 1 / 2019, GX / P2V / 2017, RaTG13, etc. sarbecovirus, and can be used for preventing and treating the infection of SARS-CoV-2 and its variant strains, SARS-CoV, WIV1, GD / 1 / 2019, GX / P2V / 2017, RaTG13, etc. sarbecovirus.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a broad-spectrum neutralizing antibody against sarbecovirus and its applications. Background Technology

[0002] Antibodies have become a powerful weapon in the prevention and control of infectious diseases due to their strong efficacy, high specificity, and low side effects. For example, Inmazeb, a triple-antibody cocktail targeting Ebola virus, was approved by the FDA in 2020, achieving a breakthrough in curbing Ebola mortality and improving survival rates. Viruses with similar genomic sequences to SARS-CoV-2 and SARS-CoV, namely sarbecoviruses such as GD / 1 / 2019, GX / P2V / 2017, and RaTG13, have been detected in multiple species. Therefore, there is an urgent need to further screen and design broad-spectrum neutralizing antibodies and develop more efficient and broad-spectrum antibody drugs.

[0003] The spike protein (S protein) on the surface of coronaviruses plays a crucial role in mediating viral invasion of cells, making it an important target for neutralizing antibody development and vaccine design. The S protein consists of two subunits, S1 and S2. S1 is responsible for receptor recognition, while S2 mediates membrane fusion. S1 is further divided into an N-terminal domain (NTD) and a C-terminal domain (CTD). The CTD, also known as the RBD, recognizes the receptor ACE2, thereby promoting viral infection. Currently, most reported neutralizing antibodies against SARS-CoV-2 target the RBD, inhibiting viral infection by blocking receptor binding. However, with the continuous emergence of SARS-CoV-2 variants, a series of mutations have occurred in the RBD region, reducing the efficacy of existing antibodies. Therefore, there is an urgent need to develop antibodies targeting the conserved regions of the S protein, including the RBD, NTD, and S2.

[0004] The purpose of this invention is to screen and design antibodies with broad-spectrum neutralizing activity against SARS-CoV-2 and its variant strains, as well as other sabeviruses. Summary of the Invention

[0005] To obtain a human monoclonal antibody with neutralizing activity, this invention first uses SARS-CoV-2 RBD expressed in mammalian cells as an antigen. Memory B cells that specifically bind to the SARS-CoV-2 RBD protein were screened from PBMCs of recovered SARS-CoV-2 patients via flow cytometry. Then, the sorted individual B cells were subjected to reverse transcription PCR and nested PCR to amplify the variable region sequence of the antibody using specific primers, and further ligated to the constant region of the antibody into an expression vector. After expression and purification of the antibody in mammalian cells, a series of functional tests were performed, including the ability to bind to the RBD protein of SARS-CoV-2 and other sabeban viruses, and the effect of neutralizing SARS-CoV-2 and other sabeban virus infections. A broad-spectrum human monoclonal antibody 74 was obtained that neutralizes SARS-CoV-2 and its variants, GD / 1 / 2019, RaTG13, SARS-CoV, WIV1, and other sabeban virus infections.

[0006] To obtain broad-spectrum antibodies with higher neutralizing activity, this invention uses the 74 antibody as a backbone and embeds three nanoantibodies previously identified by the inventors' team—R14 (patent number: 202210759496.5), R211 (patent number: 202211433688.3), and S102 (patent number: 202211433689.8)—into the 74 antibody heavy chain, constructing 10 chimeric heavy chains: 74-R14, 74-R211, 74-S102, R14-74, R211-74, S102-74, 74-R14-S102, 74-S102-R14, 74-R211-S102, and 74-S102-R211. These are then combined with the 74 antibody light chain to form 6 bispecific antibodies and 4 trispecific antibodies.

[0007] Specifically, the present invention is achieved through the following aspects.

[0008] In one aspect, the present invention provides a broad-spectrum human neutralizing antibody 74 against sabevirus or an antigen-binding fragment thereof.

[0009] Its heavy chain variable region has three complementarity-determining regions (CDRs) with amino acid sequences selected from the following group:

[0010] CDR1 as shown in SEQ ID NO:1

[0011] CDR2 as shown in SEQ ID NO:2, and

[0012] CDR3 as shown in SEQ ID NO:3;

[0013] Its three complementarity-determining regions (CDRs) of the light chain variable region have amino acid sequences selected from the following group:

[0014] As shown in SEQ ID NO:4, CDR1,

[0015] CDR2 as shown in SEQ ID NO:5, and

[0016] CDR3 as shown in SEQ ID NO:6.

[0017] In one embodiment, the neutralizing 74 antibody or its antigen-binding fragment contains:

[0018] As shown in SEQ ID NO:7, the heavy chain variable region, and

[0019] As shown in SEQ ID NO:8, the light chain variable region.

[0020] In one embodiment, the neutralizing antibody 74 or its antigen-binding fragment contains:

[0021] Heavy chains, as shown in SEQ ID NO: 9, and

[0022] Light chains as shown in SEQ ID NO:10.

[0023] In another aspect, the present invention provides six strains of broad-spectrum bispecific neutralizing antibodies against sabezia virus, which are obtained by embedding nanobodies R14, R211, and S102 into the heavy chain of the broad-spectrum neutralizing antibody 74 against sabezia virus as described in any one of claims 1 to 3 or its antigen-binding fragment. Specifically, these are 74-R14, 74-R211, 74-S102, R14-74, R211-74, and S102-74, as well as four strains of broad-spectrum trispecific neutralizing antibodies against sabezia virus 74-R14-S102, 74-S102-R14, 74-R211-S102, and 74-S102-R114. The antibodies or their antigen-binding fragments contain the heavy chain shown in SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0024] In one embodiment, the antigen-binding fragment is selected from Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, and biantibody.

[0025] In another aspect, the present invention provides a polypeptide obtained by embedding nanobodies R14, R211, and S102 into the heavy chain of the broad-spectrum neutralizing antibody 74 of the sabevirus or its antigen-binding fragment, more preferably containing a sequence selected from SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0026] In another aspect, the present invention provides a polynucleotide encoding any of the foregoing neutralizing antibodies or their antigen-binding fragments or polypeptides.

[0027] In another aspect, the present invention provides an expression vector comprising the aforementioned polynucleotides.

[0028] In another aspect, the present invention provides a host cell comprising the above-described expression vector.

[0029] In another aspect, the present invention provides a pharmaceutical composition comprising any of the foregoing neutralizing antibodies or their antigen-binding fragments and a pharmaceutical carrier.

[0030] In another aspect, the present invention provides the use of any of the above-mentioned neutralizing antibodies or their antigen-binding fragments in the preparation of medicaments for the treatment and prevention of SARS-CoV-2 and its variants, SARS-CoV, WIV1, GD / 1 / 2019, GX / P2V / 2017, RaTG13 and other sabevirus infections.

[0031] definition

[0032] "Antigen-binding fragment" refers to the antigen-binding fragment of an antibody and antibody analogues, which typically includes at least a portion of the antigen-binding region or variable region of the parent antibody, such as one or more CDRs. The antibody fragment retains at least some of the binding specificity of the parent antibody. Antigen-binding fragments include those selected from Fab, Fab′, Fab′-SH, Fv, scFv, F(ab′)2, biantibodies, and peptides containing CDRs.

[0033] “Fab” consists of a light chain, a heavy chain, a variable region, and CH1.

[0034] The “Fab′ fragment” contains a light chain and a heavy chain portion that includes the variable region and the region between the CH1 or CH1 and CH2 domains. Interchain disulfide bonds are formed between the two heavy chains of the two Fab′ fragments to form the F(ab′)2 molecule.

[0035] The “F(ab′)2 segment” contains two light chains and two heavy chains containing portions of a constant region between the CH1 and CH2 domains, thereby forming interchain disulfide bonds between the two heavy chains. Therefore, the F(ab′)2 segment consists of two Fab′ segments held together by disulfide bonds between the two heavy chains.

[0036] The “Fv region” contains variable regions from both the heavy and light chains, but lacks constant regions.

[0037] "Single-chain Fv antibody (scFv antibody)" refers to an antigen-binding fragment containing antibody variable regions, which are encapsulated within a single polypeptide chain. Generally, scFv contains a polypeptide linker between the heavy chain variable region and the light chain variable region, which allows the scFv to form the desired structure for antigen binding.

[0038] A "dual antibody" is a small antigen-binding fragment with two antigen-binding sites. The fragment contains a VH (VH-VL or VL-VH) linked to a VL in the same polypeptide chain. By using a linker so short that it cannot pair between two domains on the same chain, the domain pairs with a complementary domain of the other chain to form two antigen-binding sites.

[0039] This invention also provides pharmaceutical compositions containing a neutralizing antibody or its antigen-binding fragment that broadly neutralizes sabeziovirus according to this invention. To prepare the pharmaceutical composition, the antibody or its antigen-binding fragment can be mixed with a pharmaceutical carrier or excipient to prepare various desired dosage forms. Examples of dosage forms for the pharmaceutical compositions of this invention include, for example, oral dosage forms such as tablets, powders, pills, granules, fine granules, soft / hard capsules, film-coated tablets, small pellets, sublingual tablets, and ointments; and non-oral dosage forms such as injections, suppositories, transdermal preparations, ointments, plasters, and topical liquids. Those skilled in the art can select appropriate dosage forms based on the route of administration and the target population.

[0040] The dosage of the active ingredient in the pharmaceutical composition of the present invention varies depending on the target patient, the target organ, symptoms, method of administration, etc. It can be determined based on the doctor's judgment, taking into account the type of dosage form, method of administration, patient's age and weight, patient's symptoms, etc.

[0041] The beneficial effects of this invention are:

[0042] This invention screened and obtained a broad-spectrum human neutralizing antibody 74 against sabezia virus, and designed and obtained six broad-spectrum bispecific neutralizing antibodies against sabezia virus: 74-R14, 74-R211, 74-S102, R14-74, R211-74 and S102-74, as well as four broad-spectrum trispecific neutralizing antibodies against sabezia virus: 74-R14-S102, 74-S102-R14, 74-R211-S102 and 74-S102-R114. These neutralizing antibodies can bind to the S protein of sabezioviruses broadly, including but not limited to SARS-CoV-2 and its variants, SARS-CoV, GD / 1 / 2019, GX / P2V / 2017, RaTG13, etc., and inhibit sabeziovirus infections such as SARS-CoV-2 and its variants, SARS-CoV, GD / 1 / 2019, GX / P2V / 2017, RaTG13, etc., and have the potential to treat and prevent sabeziovirus infections including but not limited to SARS-CoV-2 and its variants, SARS-CoV, etc. Attached Figure Description

[0043] Figure 1 Molecular sieve chromatography and SDS-PAGE identification of 74 and representative bispecific antibodies (74-S102) and trispecific antibodies (74-S102-R14);

[0044] Figures 2A-2C : 74. Kinetic curves of broad-spectrum binding of antibodies to SARS-CoV-2 and its variants, SARS-CoV, GD / 1 / 2019, GX / P2V / 2017, RaTG13 and other sabevir RBDs; among which, Figure 2A The binding kinetics curves of the 74 antibody with the Prototype (PT), Alpha, Beta, Delta strains of SARS-CoV-2, as well as OmicronBA.1 and BA.1.1 are shown. Figure 2B The binding kinetics of antibody 74 with OmicronBA.2, BA.2.75, BA.4 / 5, GD / 1 / 2019, GX / P2V / 2017 and RaTG13 are shown. Figure 2C The binding kinetics curves of antibody 74 with SARS-CoV and WIV1 are shown.

[0045] Figures 3A to 3D The effects of 74, 74-R14, 74-R211, 74-S102, R14-74, R211-74, S102-74, 74-R14-S102, 74-S102-R14, 74-R211-S102, and 74-S102-R14 on neutralizing different sabeviral pseudovirus infections are shown. Figure 3AThe effects of each antibody on the broad-spectrum neutralization of the prototype strain (PT), Alpha, Beta, and Delta strains of the virus were demonstrated. Figure 3B The antibodies were shown to have broad-spectrum neutralizing effects against OmicronBA.1, BA.1.1, and BA.2 viral infections. Figure 3C The antibodies demonstrated broad-spectrum neutralizing effects against OmicronBA.2.75, BA.4 / 5, GD / 1 / 2019, and GX / P2V / 2017 viral infections. Figure 3D The antibodies demonstrated broad-spectrum neutralizing effects against RaTG13, SARS-CoV, and WIV1 viral infections. Detailed Implementation

[0046] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0047] Example 1: Isolation of SARS-CoV-2 RBD protein-specific memory B cells

[0048] With informed consent from recovered SARS-CoV-2 patients, 15 mL of blood was collected, and PBMCs were separated using lymphocyte separation tubes (purchased from Dakota). The separated PBMCs were incubated with SARS-CoV-2 RBD protein (final concentration 400 nM) on ice for 30 min, then washed twice with PBS, and subsequently incubated with the following antibodies (purchased from BD or Miltenyi): anti-human CD3 / PE-Cy5, anti-human CD16 / PE-Cy5, anti-human CD235a / PE-Cy5, anti-human CD19 / APC-Cy7, anti-human CD27 / PacificBlue, anti-human IgG / FITC, and anti-His / PE. After incubation with the antibodies on ice for 30 min, the cells were washed twice with PBS and transferred to flow cytometry tubes. PE-Cy5 was collected after FACSAria III sorting. APC-Cy7 + Pacific Blue + FITC + PE + The cell population, namely antigen-specific memory B cells, was directly collected into a 96-well plate, with one cell per well.

[0049] Example 2: Amplification of BCR sequence in a single memory B cell and construction of an IgG all-antibody expression vector

[0050] The memory B cells obtained in Example 2 were reverse transcribed using thermostable M-MVL reverse transcriptase (purchased from Beijing Jialan), and template conversion and adapter addition were performed using TSO primers at 42°C for 90 min; then at 50°C for 2 min, 42°C for 2 min, for 10 cycles; and at 70°C for 15 min to obtain cDNA.

[0051] The cDNA of this reverse transcription product was used as a template for dsDNA amplification and enrichment using HotStar Tap Plus enzyme (QIAgen). The reaction conditions were as follows: 95℃, 5 min; 95℃, 30 s, 60℃, 30 s, 72℃, 90 s; 30 cycles; 72℃, 10 min.

[0052] Using the above PCR product as a template, nested PCR was performed to specifically amplify the antibody variable region sequence. The first round of PCR (PCRa) reaction conditions were as follows: 95℃, 5 min; 95℃, 30 s, 55℃ (H chain / κ chain) or 50℃ (… (H chain), 30s, 72℃, 90s, 35 cycles; 72℃, 7min. Use this product as a template for a second round of PCR (PCRb), with the following reaction conditions: 95℃, 5min; 95℃, 30s, 58℃ (H chain) or 60℃ (κ chain) or 64℃ ( (Chain), 30s, 72℃, 90s, 35 cycles; 72℃, 7min, to obtain PCR product.

[0053] PCR products were separated by 1.2% agarose gel electrophoresis. Bands with a size of ~400 bp were excised, recovered, and sequenced. The sequences were analyzed using IgBlast or IMGT online databases.

[0054] The antibody variable region sequence obtained from the analysis was linked with the corresponding heavy / light chain constant regions via homologous recombination and cloned into the expression vector pCAGGS (preserved in the laboratory) to obtain IgG full antibody light and heavy chain recombinant expression plasmids. The full antibody design strategy is as follows:

[0055] Heavy chain: CMV promoter-EcoRI-signal peptide-heavy chain variable region (VH)-CH-Xho I;

[0056] Light chain: CMV promoter-EcoRI-signal peptide-light chain variable region (VL)-CL ( ) -Xho I.

[0057] The heavy chain amino acid sequence of the neutralizing antibody 74, as sequenced, is shown in SEQ ID NO: 9, and the light chain amino acid sequence is shown in SEQ ID NO: 10. Analysis revealed that the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8. The three complementarity-determining regions (CDRs) of the heavy chain variable region were identified as having amino acid sequences selected from the following group: CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 2, and CDR3 as shown in SEQ ID NO: 3; the three complementarity-determining regions (CDRs) of the light chain variable region had amino acid sequences selected from the following group: CDR1 as shown in SEQ ID NO: 4, CDR2 as shown in SEQ ID NO: 5, and CDR3 as shown in SEQ ID NO: 6.

[0058] SEQ ID NO:1:GYNFSRYW; SEQ ID NO:2:IYPDDSDT; SEQ ID NO:3:

[0059] ARFGAGMTGMPRYFDTTRWFDP. SEQ ID NO:4: SSNIGAGYD; SEQ ID NO:5:

[0060] GNS; SEQ ID NO:6: QSYDNDLSQV.

[0061] SEQ ID NO:7: QVQLVQSGAQLKKPGESLKISCKGSGYNFSRYWIAWVRHMPGKGLEVMGIIYPDDSDTRYSPSVRGQVTISADKSTSIVYLQWSSLKASDTGIYYCARFGAGMTGMPRYFDTTRWFDPWGQGTQVTVSS.

[0062] SEQ ID NO:8: QSVLTQPPSVSGAPGQRVTISCLGGSSNIGAGYDVHWYQHLPGAAPKLLISGNSNRPSGVPARFSGSKSGTSASLAITGLQAEDEADYYCQSYDNDLSQVFGGGTKLTVLGQPKA.

[0063] SEQ ID NO: 9: METDTLLLWVLLLWVPGSTGDQVQLVQSGAQLKKPGESLKISCKGSGYNFSRYWIAWVRHMPGKGLEVMGIIYPDDSDTRYSPSVRGQVTISADKSTSIVYLQWSSLKASDTGIYYCARFGAGMTGMPRYFDTTRWFDPWGQGTQVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0064] SEQ ID NO: 10: METDTLLLWVLLLWVPGSTGDQSVLTQPPSVSGAPGQRVTISCLGGSSNIGAGYDVHWYQHLPGAAPKLLISGNSNRPSGVPARFSGSKSGTSASLAITGLQAEDEADYYCQSYDNDLSQVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS.

[0065] Example 3: Expression and purification of 74 antibody

[0066] 293F cells were co-transfected with the recombinant expression plasmid containing the light and heavy chains of the antibody obtained in Example 2, and cultured for 3-5 days. The supernatant was collected, centrifuged at 8000 rpm for 30 min, filtered through a 0.22 μm filter, and bound to a pre-packed protein A column (5 mL, GE Healthcare). The bound antibody protein was eluted with 100 mM Gly-HCl (pH 3.0), the target protein was collected, concentrated, and subjected to molecular sieve chromatography. The antibody protein was determined by SDS-PAGE (reducing and non-reducing), and the results are shown below. Figure 1 This yielded 74 antibody protein with high purity.

[0067] Example 4: Detection of antibody-antigen binding ability using surface plasmon resonance technology

[0068] Surface plasmon resonance analysis was performed using a Biacore 8K (GE Healthcare). The specific steps are as follows:

[0069] A protein A chip (GE Healthcare) was used. The purified 74 antibody obtained in Example 3 was immobilized on the chip via protein A binding to antibody Fc. The antibody immobilization amount was approximately 500 RU. The RBD protein of SARS-CoV-2 and its variants (such as sabezioviruses) was serially diluted with a solution containing 10 mM HEPES and 150 mM NaCl (pH 7.4), and the sample was loaded onto the chip surface. Changes in the response values ​​were recorded. The kinetic curves of antibody binding to RBD were analyzed using BIAevaluation software 8K (GE Healthcare). Figures 2A to 2C As shown in Table 1, the kinetic constants of antibody binding to RBD are as follows. The results show that antibody 74 has a high affinity for RBD.

[0070] Table 1. Kinetic constants of the binding of antibody 74 to RBD proteins of SARS-CoV-2 and other sabezi viruses.

[0071]

[0072] Example 5: Detection of the neutralizing effect of antibody 74 on SARS-CoV-2 and other sabevirus pseudoviruses

[0073] The purified 74 antibody obtained in Example 3 was serially diluted 3-fold starting from 200 μg / mL, for a total of 10 dilutions. Each dilution was mixed with an equal volume of pseudoviruses such as SARS-CoV-2 containing approximately 1000 TU of fluorescence and incubated at 37°C for 1 h. The resulting solutions were then added to 96-well plates pre-coated with Vero E6 cells and cultured for approximately 20 h. Fluorescence values ​​were read using a CQ1 laser confocal high-content cell analyzer (Yokogawa), and the data were analyzed using GraphPad Prism8 software to calculate the antibody's IC50. 50 The (half-maximal inhibitory concentration) value was used to further analyze the neutralization effect. Results are as follows: Figures 3A to 3D As shown in Table 2, the statistical results are presented.

[0074] Table 2 shows the neutralizing effects of 74 and bispecific / trispecific antibodies against SARS-CoV-2 and other sabevirus pseudoviruses.

[0075]

[0076] It is evident that antibody 74 can broadly neutralize infections caused by SARS-CoV-2 and its variants, SARS-CoV, GD / 1 / 2019, GX / P2V / 2017, RaTG13, WIV1, and other sabeviruses.

[0077] Example 6: Design of Broad-Spectrum Bi / Tri-Specific Neutralizing Antibodies Against Sabevirus

[0078] To obtain a broad-spectrum antibody with higher neutralizing activity, this invention uses the 74 antibody as a backbone and inserts one of the three nanobodies R14, R211, and S102 previously identified in our laboratory between VH and CH1 or after CH3 of the 74 antibody heavy chain. For example, the sequence of nanobodies R14 is inserted between VH and CH1 of the 74 antibody heavy chain through homologous recombination to form the bispecific antibody R14-74 (amino acid sequence as shown in SEQ ID NO:14). If the sequence of R14 is linked to CH3 of the 74 antibody heavy chain through homologous recombination, 74-R14 is formed (amino acid sequence as shown in SEQ ID NO:11), and so on to form R211-74 (amino acid sequence as shown in SEQ ID NO:15), 74-R211 (amino acid sequence as shown in SEQ ID NO:12), S102-74 (amino acid sequence as shown in SEQ ID NO:16), and 74-S102 (amino acid sequence as shown in SEQ ID NO:13). If two of the three nanobodies R14, R211, and S102 are inserted between VH and CH1 or after CH3 of the 74 antibody heavy chain, for example, by inserting the sequence of nanobodies R14 between VH and CH1 of the 74 antibody heavy chain through homologous recombination, and simultaneously linking the sequence of nanobodies S102 after CH3 of the 74 antibody heavy chain, a trispecific antibody chimeric heavy chain 74-R14-S102 (amino acid sequence as shown in SEQ ID NO:17) is formed. When the positions of R14 and S102 are interchanged, 74-S102-R14 (amino acid sequence as shown in SEQ ID NO:18) is formed, and so on, forming 74-R211-S102 (amino acid sequence as shown in SEQ ID NO:19) and 74-S102-R211 (amino acid sequence as shown in SEQ ID NO:20), for a total of 10 chimeric heavy chains. When combined with the 74 antibody light chain, they formed 6 bispecific antibodies and 4 trispecific antibodies.

[0079] Example 7: Expression and purification of bi / trispecific antibodies

[0080] The expression and purification of bispecific and trispecific antibodies were similar to those of antibody 74, as shown in Example 3. Protein purification results are as follows: Figure 1 .

[0081] Example 8: Detection of the neutralizing effect of bispecific / trispecific antibodies against SARS-CoV-2 and other sabevirus pseudoviruses.

[0082] The neutralizing effect of bispecific / trispecific antibodies against SARS-CoV-2 and other Sabella virus pseudoviruses was evaluated similarly to that of antibody 74 (see Example 5), and the results are shown in Table 2.

[0083] In summary, 74, 74-R14, 74-R211, 74-S102, R14-74, R211-74, S102-74, 74-R14-S102, 74-S102-R14, 74-R211-S102, and 74-S102-R114 can serve as broad-spectrum monoclonal antibodies against SARS-CoV-2 and its variants, as well as other sabeviruses.

[0084] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A broad-spectrum neutralizing antibody 74 against sabevir or its antigen-binding fragment, characterized in that, The amino acid sequences of the three complementarity-determining regions (CDRs) of its heavy chain variable region are as follows: CDR1 shown in SEQ ID NO: 1 CDR2 shown in SEQ ID NO: 2, and CDR3 as shown in SEQ ID NO: 3; The amino acid sequences of the three complementarity-determining regions (CDRs) of its light chain variable region are as follows: CDR1 shown in SEQ ID NO: 4 CDR2 shown in SEQ ID NO: 5, and CDR3 as shown in SEQ ID NO:

6.

2. The broad-spectrum neutralizing antibody 74 against sabevirens or its antigen-binding fragment as described in claim 1, characterized in that, It contains: As shown in SEQ ID NO: 7, the heavy chain variable region, and The light chain variable region is shown in SEQ ID NO:

8.

3. The broad-spectrum neutralizing antibody 74 against sabevirens or its antigen-binding fragment as described in claim 1 or 2, characterized in that, It contains: Heavy chains, as shown in SEQ ID NO: 9, and Light chains as shown in SEQ ID NO:

10.

4. A broad-spectrum neutralizing antibody against sabevirus containing a chimeric heavy chain or an antigen-binding fragment thereof, characterized in that, It is obtained by embedding nanobodies R14, R211, and S102 into the heavy chain of the broad-spectrum neutralizing antibody 74 of sabevirus as described in any one of claims 1 to 3 or its antigen-binding fragment; the amino acid sequence of the chimeric heavy chain is shown in SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, and the amino acid sequence of the light chain of the broad-spectrum neutralizing antibody of sabevirus or its antigen-binding fragment is shown in SEQ ID NO:

10.

5. The broad-spectrum neutralizing antibody against sabevir containing a chimeric heavy chain or its antigen-binding fragment as described in claim 4, characterized in that, The antigen-binding fragment is selected from Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2 or a double antibody.

6. A polynucleotide encoding the broad-spectrum neutralizing antibody 74 against sabevirus as described in any one of claims 1-3 or its antigen-binding fragment, or the broad-spectrum neutralizing antibody against sabevirus containing a chimeric heavy chain as described in claim 4 or 5 or its antigen-binding fragment.

7. An expression vector comprising the polynucleotide of claim 6.

8. A host cell comprising the expression vector of claim 7.

9. A pharmaceutical composition comprising the broad-spectrum neutralizing antibody 74 against sabevirus as described in any one of claims 1-3 or its antigen-binding fragment, or the broad-spectrum neutralizing antibody against sabevirus containing a chimeric heavy chain as described in claim 4 or 5 or its antigen-binding fragment.

10. The pharmaceutical composition according to claim 9, characterized in that, It also includes pharmaceutical carriers.

11. The use of the broad-spectrum neutralizing antibody 74 against sabevirus or its antigen-binding fragment according to any one of claims 1-3 in the preparation of a medicament for treating and preventing viral infections, wherein the virus is sabevirus; The sarbevirus was selected from SARS-CoV-2 prototype strain, Alpha strain, Beta strain, Delta strain, BA.1 strain, BA.1.1 strain, BA.2.75 strain, BA.4 / 5 strain, GD / 1 / 2019, GX / P2V / 2017, SARS-CoV or WIV1.

12. The use of the broad-spectrum neutralizing antibody against sabevirus containing a chimeric heavy chain or its antigen-binding fragment as described in claim 4 or 5 in the preparation of a medicament for treating and preventing viral infections, wherein the virus is sabevirus; The sabevirus was selected from the SARS-CoV-2 prototype strain, Delta strain, BA.1 strain, BA.1.1 strain, GD / 1 / 2019, GX / P2V / 2017, SARS-CoV, or WIV1.

13. The use of a broad-spectrum neutralizing antibody against sabevirus containing a chimeric heavy chain or an antigen-binding fragment thereof in the preparation of a medicament for treating and preventing viral infections, wherein the virus is sabevirus; The sabevirus is RaTG13; The chimeric heavy chain amino acid sequence of the broad-spectrum neutralizing antibody against sabevirus or its antigen-binding fragment is shown in any of the following: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20; the light chain amino acid sequence of the broad-spectrum neutralizing antibody against sabevirus or its antigen-binding fragment is shown in SEQ ID NO:

10.

14. The use of a broad-spectrum neutralizing antibody against sabevirus containing a chimeric heavy chain or an antigen-binding fragment thereof in the preparation of a medicament for treating and preventing viral infections, wherein the virus is sabevirus; The sab virus is a SARS-CoV-2 Beta strain, Alpha strain, BA.2 strain, BA.2.75 strain, or BA.4 / 5 strain; The chimeric heavy chain amino acid sequence of the broad-spectrum neutralizing antibody against sabevirus or its antigen-binding fragment is any one of the following: SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20; the light chain amino acid sequence of the broad-spectrum neutralizing antibody against sabevirus or its antigen-binding fragment is shown in SEQ ID NO: 10.

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